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Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes
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Bailey, Trisha L., Hernandez-Fernaud, Juan Ramon and Gibson, Matthew I. (2021) Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes. RSC Medicinal Chemistry, 12 (6). pp. 982-993. doi:10.1039/D1MD00078K
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WRAP-proline-pre-conditioning-cell-monolayers-increases-post-thaw-recovery-viability-distinct-mechanisms-other-osmolytes-Gibson-2021.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution. Download (4008Kb) | Preview |
Official URL: http://dx.doi.org/10.1039/D1MD00078K
Abstract
Cell cryopreservation is an essential tool for drug toxicity/function screening and transporting cell-based therapies, and is essential in most areas of biotechnology. There is a challenge, however, associated with the cryopreservation of cells in monolayer format (attached to tissue culture substrates) which gives far lower cell yields (<20% typically) compared to suspension freezing. Here we investigate the mechanisms by which the protective osmolyte L-proline enhances cell-monolayer cryopreservation. Pre-incubating A549 cells with proline, prior to cryopreservation in monolayers, increased post-thaw cell yields two-fold, and the recovered cells grow faster compared to cells cryopreserved using DMSO alone. Further increases in yield were achieved by adding polymeric ice recrystallization inhibitors, which gave limited benefit in the absence of proline. Mechanistic studies demonstrated a biochemical, rather than biophysical (i.e. not affecting ice growth) mode of action. It was observed that incubating cells with proline (before freezing) transiently reduced the growth rate of the cells, which was not seen with other osmolytes (betaine and alanine). Removal of proline led to rapid growth recovery, suggesting that proline pre-conditions the cells for cold stress, but with no impact on downstream cell function. Whole cell proteomics did not reveal a single pathway or protein target but rather cells appeared to be primed for a stress response in multiple directions, which together prepare the cells for freezing. These results support the use of proline alongside standard conditions to improve post-thaw recovery of cell monolayers, which is currently considered impractical. It also demonstrates that a chemical biology approach to discovering small molecule biochemical modulators of cryopreservation may be possible, to be used alongside traditional (solvent) based cryoprotectants.
| Item Type: | Journal Article | |||||||||||||||
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| Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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| Divisions: | Faculty of Science, Engineering and Medicine > Science > Chemistry Faculty of Science, Engineering and Medicine > Medicine > Warwick Medical School |
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| Library of Congress Subject Headings (LCSH): | Cells -- Cryopreservation , Antifreeze proteins, Molecular chaperones | |||||||||||||||
| Journal or Publication Title: | RSC Medicinal Chemistry | |||||||||||||||
| Publisher: | Royal Society of Chemistry | |||||||||||||||
| ISSN: | 2632-8682 | |||||||||||||||
| Official Date: | 1 June 2021 | |||||||||||||||
| Dates: |
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| Volume: | 12 | |||||||||||||||
| Number: | 6 | |||||||||||||||
| Page Range: | pp. 982-993 | |||||||||||||||
| DOI: | 10.1039/D1MD00078K | |||||||||||||||
| Status: | Peer Reviewed | |||||||||||||||
| Publication Status: | Published | |||||||||||||||
| Access rights to Published version: | Open Access | |||||||||||||||
| RIOXX Funder/Project Grant: |
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